Chemicals and reagents

Unless stated otherwise, all chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), prepared and used according to the manufacturer’s instructions. CELLSTAR® culture 96-well ultra-low attachment (ULA) plates were obtained from Greiner Bio-One (Frickenhausen, Germany). Parenteral nutrition SmofKabiven®, trace elements concentrate Addaven®, liposoluble vitamins Vitalipid® Novum Adult, and water-soluble vitamins Soluvit® Novum, all produced by Fresenius Kabi (Kriens, Switzerland), were kindly provided by the Department of Clinical Nutrition of the Universitair Ziekenhuis Brussel (Brussels, Belgium).

Cell culture and human liver spheroid co-culture characterization

Human liver spheroid co-cultures were set up using C3A cells, a clonal derivative of the human hepatoma HepG2 cell line (CRL-10741, ATCC, Manassas, VA, USA), and LX-2 cells, an immortalized activated human hepatic stellate cell (HSC) line (SCC064, Sigma-Aldrich). C3A and LX-2 cells were cultured in minimum essential medium (MEM) (Gibco, Waltham, MA, USA) supplemented with 1 mM sodium pyruvate, 1% non-essential amino acids (Gibco), 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C, at 5% (v/v) CO2. As schematically shown in Fig. 1A, liver spheroids were generated by seeding C3A and LX-2 cells at a ratio of 10:1 (500 C3A cells/well and 50 LX-2 cells/well in a total volume of 100 μL; day 0) on 96-well ULA plates for 72 h under gentle shaking, as described previously (Dos Santos et al. 2023). Spheroid growth over time was monitored by taking images using a Nikon Eclipse Ti-S microscope (Nikon, Tokyo, Japan) with a 10 × objective. Spheroid area, sphericity, and contours were determined by manually tracing the outlines of spheroids’ edges in ImageJ (version 1.54d; National Institutes of Health, Bethesda, MD, USA) from bright-field images taken from at least 25 spheroids on day 3 and day 9 following cell seeding. The spheroid area was extracted from the corresponding built-in measure. The spheroid sphericity was defined and calculated as the ratio of the circumference of a perfect circle with an area identical to that of the spheroid and the circumference of the spheroid itself. Spheroid contours were extracted as XY-coordinates in ImageJ and overlays of individual contours were prepared in R (version 4.2.0).

Fig. 1

Generation and characterization of human liver spheroid co-cultures. A Schematic representation of the experimental setup. B Bright-field images of spheroids on day 3 (beginning of the treatment) and day 9 (end of the longest treatment); scale bar = 250 μm. C ATP content on day 3 and day 9. D Area of spheroids on day 3 and day 9. E Sphericity of spheroids on day 3 and day 9. F Overlapped contour outlining of spheroids edges on day 3 and day 9 of culture. G Relative mRNA expression levels of hepatocyte markers. H Relative mRNA expression levels of hepatic stellate cell markers. H hepatocyte, HSC hepatic stellate cell, TPN total parenteral nutrition, Tx treatment, ALB albumin, HNF1A hepatocyte nuclear factor-1 alpha, APOB apolipoprotein B, CYP7A1 cytochrome P450 7A1, SLC27A2 long-chain fatty acid transport protein 2, DES desmin, PDGFRB platelet-derived growth factor receptor beta, COL1A1 alpha-1 type I collagen, ACTA2 smooth muscle α-actin. Data are derived from three independent experiments and expressed as mean ± SEM. Statistical analysis was done using unpaired t test

Cell exposure to TPN and its single components

A sterile three-chamber bag of SmofKabiven® parenteral nutrition (1497 mL), containing amino acid solution with electrolytes (750 mL), glucose (446 mL; 2.33 M), and lipid emulsion (281 mL), as well as trace elements concentrate Addaven® (10 mL), and vitamins preparations Vitalipid® Novum Adult and Soluvit® Novum (10 mL) was prepared according to the manufacturer’s instructions and used for cell culture experiments. The TPN mixture prepared for in vitro testing reflects the quantity and ratios of all single components contained in a final mixture preparation used in clinical settings (Table 1). Starting from day 3 following cell seeding, human liver spheroid co-cultures were exposed to TPN and its components for a variable period, ranging from 24 to 144 h (Fig. 1A). MEM cell culture medium was used to prepare different concentrations of TPN and its single components.

Table 1 Concentrations of total parenteral nutrition and its components used for exposure of co-culture liver spheroidsCell viability

Human liver spheroid co-cultures were exposed to TPN and its single components for 24, 48, 72, and 144 h. Cell viability was determined by measuring the total adenosine triphosphate (ATP) content using the CellTiter-Glo® 3D Cell Viability Assay (G9682, Promega, Leiden, Netherlands) and by following manufacturer’s instructions. Briefly, 70 μL of cell culture medium per well was removed, 30 μL of Cell TiterGlo® 3D reagent was added, and the spheroids were lysed by pipetting up and down vigorously. Following a 20-min incubation at 37 °C in the dark, 50 μL per well of the mixture lysate was transferred to a white plate (Lumitrac, Greiner Bio-One GmbH, Frickenhausen, Germany) and the bioluminescence signal was measured using SpectraMax iD3 Multi-Mode Reader (Molecular Devices, San Jose, CA, USA). At least 3 human liver spheroids per treatment condition and 15 for growth assessment over time were used to measure the total ATP.

RNA extraction, cDNA synthesis, and real-time RT-qPCR analysis

Total RNA from approximately 65 human liver spheroids cultured for 3 days and 9 days (time points corresponding to the start of the treatments and the longest treatment duration, respectively) was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and quantified using the NanoDrop® ND-1000 spectrophotometer (ThermoFisher Scientific, Merelbeke, Belgium). The synthesis of complementary DNA (cDNA) was performed using 800 ng of total mRNA per sample and by means of the iScriptTM Reverse Transcription Supermix (Bio-Rad, Temse, Belgium). Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis was performed using 10 ng of cDNA, Taqman® Fast Advanced Mastermix (Applied Biosystems, Waltham, MA, USA) and Taqman probes (ALB, Hs00609411_m1; HNF1A, Hs00167041_m1; APOB, Hs00181142_m1; CYP7A1, Hs00167982_m1; SLC27A2, Hs00167982_m1; DES, Hs00157258_m1; PDGFRB, Hs01019589_m1; COL1A1, Hs00164004_m1; ACTA2, Hs00426835_g1; HPRT1, Hs02800695_m1), following manufacturer’s instructions and using StepOnePlus™ real-time PCR system (Applied Biosystems, Waltham, MA, USA). Relative mRNA expression levels were calculated according to the Pfaffl method (Pfaffl 2001). HPRT1 was used as a housekeeping gene for normalization purposes.

RNA sequencing and transcriptome analysis

Total RNA from approximately 65 human liver spheroid co-cultures treated with 1% TPN for 24 and 144 h was extracted employing the RNeasy Mini kit (Qiagen), and only the RNA samples of high quality (RNA integrity number > 7.0) as measured using the 4150 TapeStation (Agilent Technologies, Santa Clara, CA, USA) were used. Library preparation and RNA sequencing were performed with BGI Genomics (Hong Kong, China) using DNBSEQ™ sequencing technology, as previously described (Drmanac et al. 2010). Sequence reads were checked for quality and filtered using SOAPnuke software (v1.5.2; BGI Genomics) (Cock et al. 2010; Chen et al. 2018b). The filtered clean reads were aligned to the reference human genome assembly GRCh38.p13 (version GCF_000001405.39_GRCh38.p13) using HISAT2 software (v2.0.4) and mapped to the reference transcripts using Bowtie2 software (v2.2.5), and the gene expression level of each sample was calculated using RSEM (v1.2.8) (Li and Dewey 2011; Langmead and Salzberg 2012). Differential gene expression (DEG) analysis was performed using the DESeq2 analysis method (Love et al. 2014). Transcripts with a log2-transformed gene expression fold change |log2(FC)|> 0.58 and adjusted p value (Q value) < 0.05 were deemed significantly differentially expressed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to perform the enrichment analysis of DEGs (Kanehisa 2000).

Mitochondrial membrane potential detection

Liver spheroid co-cultures were exposed to TPN and its single components for 24 and 144 h. Mitochondrial membrane potential (MMP, ΔΨM) was assessed using the cationic Rhodamine 123 probe (Ex/Em = 505/534 nm), accumulating in the negatively charged mitochondrial matrix, to stain liver spheroid co-cultures, as explained elsewhere (Dos Santos et al. 2023). The dye accumulation is inversely proportional to MMP. Briefly, after 24- or 144-h exposure to TPN and its single components, 8–12 human liver spheroid co-cultures were pooled, gently disaggregated into single-cell suspension using TrypLE™ Express (Gibco, Waltham, MA, USA), and, by pipetting up and down, washed with phosphate-buffered saline (PBS) and incubated with Rhodamine 123 (1.25 μM) for 30 min at 37 °C in the dark. Subsequently, cells were washed and resuspended in PBS and analyzed using Attune Acoustic Focusing Cytometer (Life Technologies, Carlsbad, CA, USA). Measured fluorescence was used to quantify relative MMP normalized against untreated control. As a positive control, human liver spheroid co-cultures exposed to carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (20 μM) for 72 h were used (Sakamuru et al. 2016; Dos Santos et al. 2023).

ER stress detection

Liver spheroid co-cultures were exposed to TPN and its single components for 24 and 144 h. ER stress was determined using thioflavin T (ThT; Ex/Em = 440/490 nm) to quantify intracellular protein aggregates (Verwilst et al. 2019; Dos Santos et al. 2023). Following exposure to TPN and its single components for 24 or 144 h, at least eight human liver spheroid co-cultures were pooled, gently disaggregated into single-cell suspension using TrypLE™ Express, and, by pipetting up and down, washed with PBS and stained with ThT (15 μM). Following 30 min of incubation at 37 °C in the dark, the suspension of single cells was washed once, resuspended in PBS and analyzed using the Attune Acoustic Focusing Cytometer. Detected fluorescence was used to quantify relative protein aggregates, and therefore ER stress, normalized against untreated control. Thapsigargin (Tg; 10 μM) treatment of human liver spheroid co-cultures for 72 h was used as a positive control (Lindner et al. 2020; Dos Santos et al. 2023).

Intracellular reactive oxygen species detection

Intracellular reactive oxygen species (ROS) levels were measured using chloromethyl-2′,7′-dichlorofluorescein diacetate (CM-H2DCFDA; Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. In short, after 2 h of exposure to TPN and its single components, human liver spheroid co-cultures were washed with PBS and stained with CM-H2DCFDA (10 μM). Following incubation of 25 min at 37 °C in the dark, human liver spheroid co-cultures were washed with PBS and lysed using 1% Triton X-100. A 100 μL of lysate per sample was transferred to a black 96 well-plate and the fluorescence signal was measured using SpectraMax iD3 Multi-Mode Reader (Ex/Em = 492/518 nm). Fluorescence values recorded were used to quantify relative intracellular ROS against untreated control. Human liver spheroid co-cultures exposed to menadione (100 μM) for 2 h were used as a positive control for ROS assessment (Criddle et al. 2006). At least six human liver spheroids per condition were used to measure the ROS levels.

Apoptosis analysis

Liver spheroid co-cultures were exposed to TPN and its single components for 144 h. Apoptotic cells were detected using eBioscience™ Annexin V apoptosis detection kit (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. Following 144 h exposure to TPN, 12 human liver spheroid co-cultures per sample were pooled, washed with PBS, and gently disaggregated into single-cell suspension using TrypLE™ Express and by pipetting up and down. Subsequently, cells were washed with binding buffer and stained with FITC-conjugated annexin V. Following a 15-min incubation at room temperature in the dark, cells were washed with binding buffer, stained with propidium iodide (PI), and analyzed using Attune Acoustic Focusing Cytometer. Analyzed cells were expressed as a percentage of apoptotic cells (Annexin V+PI−/+). Human liver spheroid co-cultures exposed to camptothecin (CPT; 10 μM) for 48 h were used as a positive control for apoptosis assessment (Traganos et al. 1996; Dos Santos et al. 2023).

Intracellular lipid accumulation detection using flow cytometry

Liver spheroid co-cultures were exposed to TPN and its single components for 24 and 144 h. Intracellular lipid accumulation in human liver spheroid co-cultures was quantified using BODIPY™ 493/503 neutral lipid dye and flow cytometry (Boeckmans et al. 2020). Briefly, following 24 or 144 h of exposure to TPN and its single components, at least eight human liver spheroid co-cultures were pooled, gently disaggregated into single-cell suspension using TrypLE™ Express, and, by pipetting up and down, washed with PBS and stained with BODIPY™ 493/503 neutral lipid dye (Thermo Fisher Scientific, Merelbeke, Belgium) (2 μM). Following 10 min of incubation at 37 °C in the dark, the suspension of single cells was analyzed using the Attune Acoustic Focusing Cytometer. Detected fluorescence was used to quantify intracellular lipid accumulation, normalized against untreated control. Spheroids treated with valproic acid sodium salt (VPA; 5 mM) for 48 h were used as a positive control (Bai et al. 2017).

Fluorescent staining of intracellular lipids

Liver spheroid co-cultures were exposed to either TPN (5%) or lipid emulsion (0.95%) for 144 h and VPA (5 mM) for 48 h. To stain intracellular lipids, at least six human liver spheroid co-cultures per sample were pooled and washed briefly with PBS. Subsequently, human liver spheroid co-cultures were fixed with 4% (w/v) paraformaldehyde for at least 30 min at room temperature and thoroughly washed with PBS for 5 min. Next, spheroids were quickly embedded into a PolyFreeze tissue freezing medium and stored for 2 h at − 20 °C. Spheroid-containing solid frozen blocks were then sliced using a cryotome (MICROM GmbH HM 525, Walldorf, Germany). The obtained sections of 16 μm thickness were placed onto a microscope slide (SuperFrost®Plus, VWR, Leuven, Belgium) and immediately used for fluorescent staining. Following a short washing with PBS (5 min), sections were incubated with 2 μM BODIPY™ 493/503 neutral lipid dye (Thermo Fisher Scientific, Merelbeke, Belgium) for 15 min at room temperature in the dark, washed with PBS for 5 min, and imaged using a Nikon Eclipse Ti-S microscope with a 10 × objective.

Data analysis

All data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test or an unpaired t test. A p value < 0.05 was considered significant. GraphPad Prism (version 10.1.1; GraphPad Software Inc., La Jolla, CA, USA) was used for statistical analysis. All experiments were repeated independently at least three times.